【作者】 于欢；

【导师】 王敦；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2012， 硕士

【摘要】 本研究利用澳洲雪漏斗蜘蛛毒素基因ω-ACTX-Ar1b（Ar1b）替换棉铃虫核型多角体病毒（HearNPV）的人工基因组（HZ8-bacmid）中egt基因获得的Ar1b-bacmid，通过转座将多角体基因（ph）修复到Ar1b-bacmid和HZ8-bacmid上，构建成了具有感染活性的重组病毒Ar1b-HearNPV和基因修复型对照G4株HearNPV（HZ8-HearNPV）。将二者分别转染HzAM1细胞后，在细胞水平上对比研究了Ar1b毒素基因重组的病毒与正常病毒的复制与病毒生长情况，并与野生型HearNPV G4株病毒（wt-HearNPV）对照分别感染棉铃虫幼虫，对比研究了重组病毒Ar1b-HearNPV的杀虫效果。另外，研究了野生型HearNPV感染棉铃虫后对试虫ALP活性与基因表达的影响，取得的主要结果如下：1、通过转座将ph基因修复到Ar1b-bacmid和HZ8-bacmid上，成功构建了多角体恢复型Ar1b-HearNPV和HZ8-HearNPV人工基因组。人工基因组通过转染HzAM1细胞，证实了Ar1b-HearNPV和HZ8-HearNPV均能包装成正常的病毒粒子，且二者具有相似的病毒生长曲线，但Ar1b-HearNPV较HZ8-HearNPV的病毒DNA复制能力稍低，差异不显著。2、重组病毒LD50和LD90生测结果显示：Ar1b-HearNPV相比HZ8-HearNPV和wt-HearNPV具有更高的杀虫毒力； LT50和LT90结果表明： Ar1b-HearNPV相比HZ8-HearNPV和wt-HearNPV具有更快的杀虫速率。3、不同剂量感染不同龄期幼虫结果表明：高剂量（120PIBs/larva）下，Ar1b-HearNPV与wt-HearNPV和HZ8-HearNPV导致的幼虫死亡率一致，且杀虫速度最快；低剂量（30PIBs/larva）下，Ar1b-HearNPV依旧有最快的杀虫速度，只是高龄幼虫的死亡率低于wt-HearNPV和HZ8-HearNPV处理组。4、不同龄期幼虫在不同处理下日均累积增重量测试结果表明，Ar1b-HearNPV对不同龄期的幼虫均有很好的抑制生长效果；不同龄期幼虫不同处理下日均取食量结果表明：Ar1b-HearNPV喂食的幼虫，不同龄期幼虫日均取食量均为最低的。由此可见，重组病毒能抑制幼虫的取食，同时也能抑制幼虫的生长。幼虫日累积取食总量比较结果表明：高龄幼虫被野生型病毒感染后导致幼虫累积取食量高出健康试虫；而Ar1b-HearNPV感染的幼虫，不论感染幼虫的龄期大小，取食量均显著低于两种对照病毒。5、wt-HearNPV感染后棉铃虫中肠ALP活性研究结果表明：wt-HearNPV病毒感染棉铃虫后，引起宿主昆虫中肠ALP活性显著降低，且随着病毒感染剂量的增加、或病毒感染后时间的推移ALP活性被抑制加重。wt-HearNPV感染后棉铃虫中肠ALP表达分析结果说明：病毒的感染能抑制ALP的表达。感染后时间越长，表达量越低；ALP被抑制的时间随感染剂量的增加而提前。更多还原

【Abstract】 In this study, A per os infectivity recombined HearNPV, Ar1b-HearNPV, was constructedby repair the polyhedra gene (ph) to Ar1b-bacmid and HZ8-bacmid. The recombinantHearNPV was identified as an improved recombinant through bioassays. The virulence, dailycumulative weight gain and daily diet consumption from different instars larvae infected byAr1b-HearNPVwas investigated. And the differences of ALP activity and ALP expressionprofile under HearNPV infection to host larvae were studied also, following were the majorcollusions:The ph with its own promoter in the Ha.p-ph-HTb vector was repaired to Ar1b-bacmidand HZ8-bacmid facilitated by helper plasmid. The Ar1b-HearNPV and HZ8-HearNPV OBscan be found after transfection to HzAM1cell lines using, respectively. The viral growthcurve indicated that Ar1b-HearNPV could produce BVs as well as HZ8-HearNPV. The viralDNA replication curve showed that Ar1b-HearNPV DNA had less copies than HZ8-HearNPVhad in HzAM1cell lines.The lethal dose tests showed that the Ar1b-HearNPV owend a higher virulence thanHZ8-HearNPV. The lethal time tests indicated that the Ar1b-HearNPV can speed death oflarvae.The bioassay results of2nd,3rdand4thinstars larvae infected with high dose (120PIBs/larva) and low dose (30PIBs/larva) demonstrated that the high dose of Ar1b-HearNPVhad a similar final mortality to HZ8-HearNPV and wt-HearNPV infection. For larvae thatinfected with the low dose, Ar1b-HearNPV could kill the larvae much quicker thanHZ8-HearNPV and wt-HearNPV, although the HZ8-HearNPV and wt-HearNPV treatmentsobtained a higher final mortality.The daily cumulative weight gain of different instar larvae showed that Ar1b-HearNPVcan restrain the development of the infectec larvae. On the other hand, compared withwt-HearNPV and HZ8-HearNPV infections, Ar1b-HearNPV infected larvae had the lowestdaily diet consumption regardless the larvae instars, while CK owned the highest one. Totalaccumulation of diet consumption analysis illustrated that Ar1b-HearNPV had the lowest dietconsumption. This kind of inhibition on diet consumption would be deceased with the rise of the larvae’s instars.The enzyme activity and gene expression level were determined at different stages afterviral infection with different infection titer. The results indicated that the ALP activity and itsgene expression level were inhibited significantly by HearNPV infection and this inhibitionstrength was co-related with viral infection titer and the time post infection. The viral invasionto host cell possibly could be facilitated by inhibition on ALP activity by virus. One of themechanisms of ALP activity inhibition by viral infection was down regulation of ALPexpression by virus.更多还原